Background: Anomalous development of the ureterovesical junction (UVJ), which joins the ureter to the bladder, is associated with vesicoureteral reflux (VUR), the abnormal retrograde flow of urine from bladder towards the kidneys. VUR is prevented by the muscular layer of the ureters and the bladder that produce a valve-like effect that occludes the UVJ during voiding. Human studies have demonstrated that refluxing UVJs have elevated levels of fibrillary collagens, and atrophy and degeneration in the ureteral smooth muscle compared to non-refluxing ureters, suggesting that the integrity of the ECM is crucial for preventing VUR at the UVJ. Longitudinal clinical studies of young children with VUR have demonstrated a 50%-65% rate of spontaneous resolution of VUR. This could be due to changes in the ECM of the UVJ that result in resolution of VUR. A large kindred was recently discovered in which patients with VUR and joint hypermobility-type Ehlers Danlos Syndrome (JHEDS) have heterozygous missense mutations (G1331R, T3257I) in Tenascin X (TNXB), an extracellular matrix glycoprotein that regulates collagen and elastin deposition through its FNIII domain, which is also implicated in the activation of latent TGF-β. Aside from joint laxity, these individuals also have hyperextensible and fragile skin, suggesting a global derangement in ECM composition.

Hypothesis: Perturbed expression of fibrillar collagens, elastin, and TNXB leads to anomalous development of the UVJ, making it susceptible to VUR.

Results: TNXB immunohistochemistryand collagen staining were performed on urinary tract sections of newborn and adult mice from the non-refluxing C57Bl/6J inbred mouse line. Abundant collagen expression is observed in the lamina propria of the UVJ of newborn and adult mice, and in the bladder muscle surrounding the UVJ in adult mice. Comparison between adult and newborn mice shows an increase in the amount of collagen deposited in the lamina propria of the bladder and the UVJ over time, suggesting that the ECM changes during the urinary tract development. TNXB expression was observed throughout development in both refluxing inbred C3H mice and in non-refluxing B6 mice. TNXB is expressed ubiquitously in the urothelium as well as the lamina propria and smooth muscle layer of the lower urinary tract. TNXB knockout mice were generated: these mice have hyperextensible skin, but they do not exhibit reflux. The TNXB gene was sequenced in 96 children with VUR. Sequencing results shows that patients in our cohort do not specifically have the G1331R or T3257I mutations, however we have identified novel missense, and deleterious mutations in the FNIII domain of TNXB. These patients are currently being tested for JHEDS using the Beighton scoring system.

Conclusions: Our results suggest there is a link between ECM perturbations and VUR.